Modulating regulatory t cell activity via interleukin 35

ABSTRACT

Methods for regulating T cell function in a subject, particularly regulatory T cell activity are provided. Methods of the invention include administering to a subject a therapeutically effective amount of an Interleukin 35-specific binding agent, such as an antibody or small molecule inhibitor. The invention further provides methods for enhancing the immunogenicity of a vaccine or overcoming a suppressed immune response to a vaccine in a subject, including administering to the subject a therapeutically effective amount of an IL35-specific binding agent and administering to the subject a vaccine. In one embodiment the vaccine is a cancer vaccine.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 12/441,166, filedAug. 7, 2009, which was a national stage filing under 35 U.S.C. 371 ofPCT/US07/079,310, filed Sep. 24, 2007, which International Applicationwas published by the International Bureau in English on Mar. 27, 2008,and which claims the benefit of U.S. Application 60/846,434, filed Sep.22, 2006, each of which are hereby incorporated herein in its entiretyby reference.

RECOGNITION OF RESEARCH FUNDING

This invention was supported by funds received from the AmericanLebanese Syrian Associated Charities (ALSAC).

FIELD OF THE INVENTION

The present invention relates to methods for regulating T cell functionin a subject, particularly regulatory T cell activity.

BACKGROUND OF THE INVENTION

The Epstein-Barr virus-induced gene 3 (EBI3; IL27b) product is a novelsoluble hematopoietin component related to the p40 subunit (IL12b) ofInterleukin 12 (IL12). EBI3 is widely expressed in cells and accumulatesin the endoplasmic reticulum and associates with the molecular chaperonecalnexin. Besides promoting Th1 cytokine production, EBI3 plays acritical regulatory role in the induction of Th2-type immune responsesand the development of Th2-mediated tissue inflammation in vivo, whichmay be mediated through the control of invariant natural killer (NK) Tcell function.

Interleukin 12 was identified and purified from the cell culture mediaof Epstein-Barr virus (EBV)-transformed B lymphoblastoid cell lines.Interleukin 12 is a 70 kDa heterodimeric cytokine composed of twodisulfide-linked glycoproteins, p40 and p35 (IL12a). Interleukin 12 isprimarily produced by macrophages and other antigen-presenting cells.Interleukin 12 has pleiotropic effects in the development of Th1responses in NK and T lymphocytes, including induction of interferon(INF)-γ production, proliferation, and enhancement of cytotoxicactivity, and inhibits Th2 responses.

Multiple, complex and interconnecting mechanisms control discriminationbetween self and non-self, including the thymic deletion of autoreactiveT cells and the induction of anergy in peripheral T cells. In additionto these passive mechanisms, active suppression of autoreactiveresponder T cells is mediated by regulatory or suppressor T cells.Regulatory T (T_(R)) cells are powerful inhibitors of T cell activationboth in vivo and in vitro. Regulatory T cells inhibit autoimmunity andinflammation, maintain immunologic tolerance, and are involved in theinduction of tumor antigen tolerance (for reviews, see, Shevach, E. M.,Nat. Rev. Immunol. 2:389-400, 2002; Sakaguchi, S., Ann. Rev. Immunol.22:531-562, 2004; and Mapara and Sykes, J. Clin. Oncology 22:1136-51,2004).

A major factor limiting immune recognition of cancer cells is the factthat tumors arise from a subject's own tissue and therefore expressmainly self antigens to which the subject's T cells have been tolerized,either centrally (i.e., in the thymus) or peripherally. This situationis manifested as tolerance of T cells that display a high avidity forthe normal self antigens expressed by the tumor, leaving only functionalT cells with low avidity. This problem is exemplified by p53. Because ofits high level of expression in certain malignancies, wild-type p53 is apotential target antigen for immunotherapy in a broad spectrum ofneoplastic diseases. However, because of low-level expression in normaltissues, T cell tolerance by clonal deletion of high-avidity T cells inthe thymus is an obstacle to generating an effective immune responsefollowing vaccination with a wild-type p53 antigen (Theobald et al., J.Exp. Med. 185:833-41, 1997). Nevertheless, it is possible to detect andclonally expand T cells specific for tumor-associated antigens (TAA)from tumor-bearing subjects. However, even if TAA-specific cells arepresent at detectable levels in tumor-bearing subjects, they are oftenincompetent to reject the tumor (Lee et al., Nat. Med. 5:677-85, 1999).

A number of vaccination approaches are currently being evaluated inclinical trials in efforts to induce host immune responses against avariety of solid tumors (e.g., colon cancer, prostate cancer, melanoma,and renal cell carcinoma). These strategies are all based on theobservation that tumors are often poor antigen presenting cells. Thelack of costimulatory molecules on their surface and the failure toproduce stimulatory cytokines may make them poorly immunogenic andsometimes even tolerogenic. The approaches investigated include the useof gene-modified tumor cells (Soiffer et al., Proc. Natl. Acad. Sci. USA95:13141-46, 1998), the use of professional antigen presenting cells(e.g., dendritic cells) or dendritic cells fused to tumor cells (Gong etal., Blood 99:2512-17, 2002; Gong et al., Nat. Med. 3:558-61, 1997), andDNA transfer using naked DNA or viral vectors.

Vaccination with dendritic cells has led to systemic T cell responses intreated subjects. However, clinical responses have been less striking,although some patients showed significant antitumor responses, includingsome complete responses (Nestle et al., Nat. Med. 4:328-32, 1998; Tjoaet al., Prostate 40:125-29, 1999; Murphy et al., Prostate 39:54-59,1999). Therefore, there remains a need for the development of effectivetherapies for enhancing antitumor immunity.

SUMMARY OF THE INVENTION

The present invention is directed to methods for inhibiting a regulatoryT cell function in a subject. In one embodiment, methods of theinvention include administering to the subject a therapeuticallyeffective amount of an Interleukin 35 (IL35; previously designatedInterleukin 34, IL34)-specific binding agent. Interleukin 35-specificbinding agents include antibodies, such as monoclonal antibodies, orfragments thereof, modified polypeptides designed to interfere with IL35formation or activity, or small molecule inhibitors, such as chemicalcompounds.

A method for treating a subject having a cancer with a cancer vaccine isalso provided. The method includes (i) administering to the subject atherapeutically effective amount of an IL35-specific binding agent and(ii) administering to the subject a cancer vaccine, where theIL35-specific binding agent enhances the efficacy of the cancer vaccine.In specific, non-limiting examples, the IL35-specific binding agentincludes an antibody, such as a monoclonal antibody, or fragmentsthereof, or a small molecule inhibitor, such as a chemical compound. Inone embodiment, administration of the therapeutically effective amountof the IL35-specific binding agent and administration of the cancervaccine is sequential, in any order. Alternatively, administration ofthe therapeutically effective amount of the IL35-specific binding agentand administration of the cancer vaccine is simultaneous.

Methods for enhancing the immunogenicity of a vaccine or overcoming asuppressed immune response to a vaccine in a subject are furtherprovided. These methods include (i) administering to the subject atherapeutically effective amount of an IL35-specific binding agent and(ii) administering to the subject a vaccine, where the IL35-specificbinding agent enhances the immunogenicity of the vaccine or overcomesthe suppressed immune response to the vaccine. In specific, non-limitingexamples, the IL35-specific binding agent includes an antibody, such asa monoclonal antibody, or fragments thereof, or a small moleculeinhibitor, such as a chemical compound. In one embodiment,administration of the therapeutically effective amount of theIL35-specific binding agent and administration of the vaccine issequential, in any order. Alternatively, administration of thetherapeutically effective amount of the IL35-specific binding agent andadministration of the vaccine is simultaneous.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that EBI3 and p35 (IL12a) are highly expressed byT_(R) cells. Real-time RT-PCR analysis of IL12-related genes wasperformed on T cells sorted from C57BL/6 mice. Data presented asrelative mRNA expression.

FIGS. 2A-E illustrate T_(R)-restricted expression of EBI3 and IL12a.Effector T (T_(E)) or T_(R) cells from the spleens and lymph nodes ofC57BL/6, Foxp3^(gfp) or EBI3^(−/−) mice were purified by FACS asindicated. FIGS. 2A & 2B. RNA was extracted and cDNA generated.Quantitative real-time PCR analysis was performed using β-actin as anendogenous control. Relative mRNA expression was determined by thecomparative CT method (* p=0.008, ** p=0.06). Data represent themean±SEM of 4 (FIG. 2A) and 2 (FIG. 2B) independent experiments. FIG.2C. Sorted T_(E) and T_(R) cells (3×10⁶ cells/lane) were cultured for 36hours in the absence of stimuli. Cells were lysed and supernatantcollected for overnight IP with an anti-IL12a (p35) mAb, eluted proteinsresolved on an SDS-PAGE gel and blotted with anti-EBI3 mAb. Two exposuretimes are shown. Data are representative of 2 independent experiments.FIG. 2D. Relative mRNA expression was determined from purified T_(E) orT_(R) cells under indicated conditions; unstimulated, stimulated for 48hours with anti-CD3/CD28 or activated in culture containing both T_(E)and T_(R) cells. Data represent the mean±SEM of 2 independentexperiments (* p=0.008). FIG. 2E. 6.5 (TCR transgenic-hemagglutininspecific) CD4⁺ T_(E) cells were purified by MACS and activated withanti-CD3/CD28 for 2 days. T cells were retrovirally transduced withvector alone or Foxp3. After resting, qPCR was performed as describedherein. Data represent the mean±SEM of 2 independent experiments (*p=0.002, ** p=0.02).

FIG. 3A-C demonstrate that EBI3 and p35 (IL12a) are required for optimalT_(R) cell function. FIG. 3A. Splenic T_(E) cells (2.5×10⁴) wereincubated with irradiated splenocytes as antigen-presenting cells(2.5×10⁴) and T_(R) cells as indicated in the presence of anti-CD3 mAb(2C11) for 60 hours, pulsed with [³H]thymidine for 8 hours and cellproliferation measured. FIG. 3B. T_(E) and T_(R) cells were sorted fromspleens and lymph nodes of wild-type (WT), EBI3^(−/−) and IL12a^(−/−)mice. Sorted T_(R) cells were mixed at different ratios withantigen-presenting cells, naïve wild-type T_(E) cells (2.5×10⁴cells/well) and 5 μM anti-CD3. Cells were cultured for 72 hours andpulsed with [³H]-thymidine (1 μCi/well) for the last 8 hours of culture.Data represent mean±SEM of 5 (4 for IL12a^(−/−) T_(R)) independentexperiments (* p=0.0002, ** p=0.008). FIG. 3C. Wild-type T_(E) cells(2×10⁶) alone or with WT, EBI3^(−/−) or IL12a^(−/−) T_(R) cells (5×10⁵)were injected intravenously into RAG1^(−/−) mice. Seven dayspost-transfer the mice were sacrificed and splenic T cell numbersdetermined by flow cytometry. Data represent mean±SEM of 3 independentexperiments with 8-12 mice per group (* p=0.002, ** p=0.02).

FIGS. 4A-B illustrate that EBI3^(−/−) T_(R) cells fail to treatinflammatory bowel disease (IBD). RAG1^(−/−) mice receivedCD4⁺CD25⁻CD45RB^(hi)T_(E) cells via the tail vein. After 3-4 weeks, micedeveloped clinical symptoms of IBD and were given a second transfer ofwild-type or EBI3^(−/−) T_(R) cells. FIG. 4A. Percent weight changefollowing T_(R) cell transfer. FIG. 4B. Colonic histology scores ofexperimental mice are shown. Data in both panels represent mean±SEM of8-11 mice per group from 4 independent experiments (* p=0.02, **p=0.05).

FIGS. 5A-C demonstrate that ectopic expression of IL35 and recombinantIL35 suppress T_(E) cell proliferation. FIG. 5A. Naïve splenic T cellswere activated for 48 hours with anti-CD3 mAb prior to transduction withEBI3, p35 (IL12a), EBI3+p35 (IL35), or pMIG (vector control). Followingtransduction, cells were expanded for 6 days, rested for 2 days andsorted for equal expression of the constructs. The T cells were thentested for their ability (at indicated cell numbers) to suppressproliferation of T_(E) cells activated with irradiated splenocytes asantigen-presenting cells (2.5×10⁴) and T_(R) cells as indicated in thepresence of anti-CD3 mAb. Effector T cells were allowed to proliferatefor 60 hours, then were pulsed with [³H]thymidine for 8 hours and cellproliferation measured. FIG. 5B. 6.5 (TCR transgenic-HA specific) CD4⁺T_(E) cells were purified by MACS and activated with anti-CD3/CD28 for 2days. T cells were retrovirally transduced, sorted and titrated into anin vitro T_(R) assay with antigen-presenting cells, 10 μg/ml HA 110-120peptide and naïve 6.5 CD4⁺CD25⁻ T_(E) cells. Data represent mean±SEM of3 independent experiments. FIG. 5C. HEK293T cells were transientlytransfected with empty GFP encoding vector or vectors containing“native” or “single chain” IL35. Cells were sorted for equivalent GFPexpression and cultured for 36 hours to facilitate protein secretion.Dialyzed, filtered supernatant from cells was mixed at indicated ratioswith anti-CD3/CD28 coated sulfate latex beads and T_(E) cells in aproliferation assay. Data represent mean±SEM of 4 independentexperiments.

DETAILED DESCRIPTION OF THE INVENTION

Compositions and methods for modulating T cell function in a subject areprovided. The compositions comprise antagonists that are specific forIL35, or the IL35 subunits EBI3 and p35 (IL12a), but do not recognizeother cytokines or cytokine combinations (e.g., an IL35-specific bindingagent). In particular, the antagonists of the invention do not recognizeor bind IL12, IL27, and the like. By “specific binding agent” isintended an agent that binds substantially only to a defined target.Thus an IL35-specific binding agent binds substantially only to asubunit (i.e., EBI3 or p35) of the heterodimeric glycoprotein or to theheterodimer itself, or inhibits IL35 activity. Likewise, an IL35receptor (IL35R)-specific binding agent binds substantially only theIL35 receptor. As IL35 shares subunits with IL12 (p35) and IL27 (EBI3),an IL35-specific binding agent that binds substantially only to IL35 butnot to IL12 or IL27 is preferred. Specific binding agents include, butare not limited to, antibodies, proteins that are designed to interferewith IL35 binding, formation or activity, proteins that compete withbinding of a subunit (i.e., EBI3 or p35) to its complement subunit,proteins that bind IL35, and small molecules. A binding agentspecifically binds if it binds only to EBI3, p35, or IL35, or fragmentsand closely related variants that share at least 80%, at least 90%, atleast 95% or greater sequence identity to EBI3, p35, or IL35.

For purposes of the present invention, percent sequence identity isdetermined using the Smith-Waterman homology search algorithm using anaffine gap search with a gap open penalty of 12 and a gap extensionpenalty of 2, BLOSUM matrix of 62. The Smith-Waterman homology searchalgorithm is taught in Smith and Waterman (Adv. Appl. Math. 2:482-489,1981). A variant may, for example, differ from the reference protein byas few as 1 to 15 amino acid residues, as few as 1 to 10 amino acidresidues, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 aminoacid residue.

By “proteins that compete with binding of a subunit” is intended aprotein that is designed to compete with binding of a subunit EBI3 orp35) to its complement subunit. In this manner, EBI3 and p35 modifiedproteins can be made that are capable of binding to the complementsubunit but that result in a defective IL35 molecule. By “modified EBI3or p35 protein” is intended an amino acid sequence for EBI3 or p35 thathas been modified by amino acid substitutions, deletions, additions andthe like. That is, the resulting IL35 molecule does not retain theimmunoregulatory activity. In this manner, mutations can be introducedinto the EBI35 or p35 amino acid sequences and the resulting proteinstested for their abilities to bind their complement subunit. Suchmodified proteins can be made recombinantly, by proteolytic digestion,by chemical synthesis, etc. Internal or terminal fragments of apolypeptide can be generated by removing one or more nucleotides fromone end or both ends of a nucleic acid which encodes the polypeptide.Mutations can be made in the corresponding nucleic acid sequenceencoding the EBI35 or p35 polypeptide and expression of the mutagenizedDNA produces modified polypeptide fragments or proteins. EBI3 and p35are known in the art. The human EBI3 gene encodes a protein of about 33kDa. The protein shares about 27% sequence identity to the p40 subunitof human IL12. Nucleic acid and amino acid sequences for EBI3 are known.See, for example, SEQ ID NOs: 1 and 2 of WO97/13859 (human) and GenBankAccession Numbers NM015766 and BC046112 (mouse). Nucleic acid and aminoacid sequences for p35 are also known in the art and include SEQ IDNOs:3 and 4 of WO97/13859 (human) and GenBank Accession NumbersNM_(—)000882 and M86672 (mouse).

Interleukin 35 refers to any intramolecular complex or single moleculecomprising at least one EBI3 polypeptide component and at least one p35polypeptide component. Typically, in vivo, EBI3 and p35 associate vianon-covalent association. For purposes of the present invention, theEBI3-p35 components may be associated with one another either covalentlyor non-covalently for the purpose of raising specific antibodies. Insome examples, EBI3 and p35 can be coexpressed as a fusion protein.

By “small molecule inhibitor” is intended a molecule of a sizecomparable to those molecules generally used in pharmaceuticals. Theterm excludes biological macromolecules (e.g., proteins, nucleic acids,etc.). Preferred small organic molecules range in size up to about 5000Da, more preferably up to 2000 Da, and most preferably up to about 1000Da. Small molecule inhibitors can disrupt protein-protein interactionsbetween a protein (both membrane bound and soluble) and its receptor,such as between the IL35 heterodimer and its receptor. The preparationof small molecule inhibitors is well known in the art. For example,although protein-protein interactions occur over a large surface area,X-ray crystallography and site-directed mutagenesis can be used to mapthe compact, centralized regions of protein-protein interfaces, oftentermed “hot spots,” that are crucial for the interaction.

Non-limiting examples of small molecule inhibitors include chemicalcompounds, inorganic molecules, organic molecules, organic moleculescontaining an inorganic component, molecules including a radioactiveatom, synthetic molecules, and peptidomimetics (e.g., short, peptidefragments that mimic the most common peptide motifs, such as an α-helixor β-sheet). As a specific binding agent, small molecule inhibitors maybe more permeable to cells, less susceptible to degradation, and lessapt to elicit an undesired immune response than large molecules.

The present invention further provides methods for inhibiting aregulatory T cell function in a subject. T_(R) cells, also known assuppressor T cells, down-regulate immune responses for both foreign andself antigens. Regulatory T cells have immunoregulatory properties andare actively involved in maintaining immune tolerance (i.e., inpreventing autoimmunity), but also control various immune reactions(Chatila, T. A., J. Allergy Clin. Immunol. 116:949-59, 2005; Bluestoneand Tang, Curr. Opin. Immunol. 17:638-42, 2005; and Schwartz, R. H.,Nat. Immunol. 6:327-30, 2005). One class of T_(R) cells,CD4⁺CD25⁺suppressor T cells, is characterized by the expression of CD4and CD25 (the Interleukin 2 receptor α-chain). These cells are oftenreferred to as “natural regulatory T cells” (Bluestone and Abbas, Nat.Rev. Immunol. 3:253-57, 2003) or “innate regulatory T cells” (Cortez etal., Transplantation 77:S12-15, 2004), and are produced by the thymus asa functionally distinct subpopulation of T cells. Their developmentcritically depends on expression of the forkhead transcription factorFoxp3 (Hori and Sakaguchi, Microbes Infect. 6:745-51, 2004). CD4⁺Foxp3⁺T_(R) cells are powerful inhibitors of T cell activation both in vivoand in vitro.

Other classes of regulatory T cells with diverse phenotypes and antigenspecificities have been described (Maggi et al., Autoimmun. Rev.4:579-586, 2005 and Levings and Roncarolo, Curr. Topics Micro. Immunol.293:303-26, 2005). For example, “adaptive regulatory T cells,” which arealso referred to as “acquired regulatory T cells,” are a population ofantigen-induced regulatory T cells induced in the periphery afterencounter with pathogens and foreign antigens (Cortez et al.,Transplantation 77:S12-15, 2004; Mills and McGuirk, Seminars Immunol.16:107-17, 2004; and Vigouroux et al., Blood 104:26-33, 2004).

By “inhibiting a regulatory T cell function in a subject” is intendedreducing and/or blocking of one or more of the suppressive effectsmediated by T_(R) cells. While not being bound by any theory, it isbelieved that T_(R) cells mediate their suppressive effects through bothcell contact-dependent mechanisms (involving their T cell receptorsand/or other cell surface-expressed molecules), and cytokine-dependentmechanisms (including, e.g., IL10 and TGF-β). In one embodiment,reducing and/or blocking of one or more of the suppressive effectsmediated by T_(R) cells is achieved by inhibiting the activation and/orproliferation of T_(R) cells. The inhibition of the activation and/orproliferation of T_(R) cells can be measured relative to a controlpopulation of cells, such as responder or effector T cells. For purposesof the invention, T_(R) cell function is reduced at least 30%, at least50%, at least 60%, at least 70%, at least 80%, or at least 90% ascompared to control cells, such as responder T cells.

As used herein, “responder T cells” or “effector T cells” refers to asubpopulation of mature T cells that facilitate an immune responsethrough cell activation and/or the secretion of cytokines. In oneembodiment, the responder T cells are CD4⁺CD25⁻ T cells. In anotherembodiment, the responder T cells are CD8⁺CD25⁻ T cells. One example ofa responder T cell is a T lymphocyte that proliferates upon stimulationby an antigen, such as a tumor antigen. Another example of a responder Tcell is a T lymphocyte whose responsiveness to stimulation can besuppressed by T_(R) cells.

Production of Anti-IL35 Antibodies

As noted herein, the invention includes antibodies specifically reactivewith IL35, EBI3 or p35. Antibodies, including monoclonal antibodies(mAbs) can be made by standard protocols. See, for example, Harlow andLane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999.Briefly, a mammal such as a mouse, hamster or rabbit can be immunizedwith an immunogenic form of a peptide. Techniques for conferringimmunogenicity on a protein or peptide include conjugation to carriersor other techniques, well known in the art. In preferred embodiments,the subject antibodies are immunospecific for antigenic determinants ofEBI3, p35, or IL35. See, SEQ ID NOs:1-4 of WO97/13859 for the humannucleic acid and amino acid sequences for EBI3 and p35, respectively,and GenBank Accession Numbers NM015766, BC046112, NM_(—)000882, andM86672 for the mouse nucleic acid and amino acid sequences for EBI3 andp35, respectively.

The antibodies of the invention include antibodies that specificallybind IL35, EBI3 and p35. As discussed herein, these antibodies arecollectively referred to as “anti-IL35 antibodies”. Thus, by “anti-IL35antibodies” is intended antibodies specific for IL35, antibodiesspecific for EBI3 and antibodies specific for p35. All of theseantibodies are encompassed by the discussion herein. The respectiveantibodies can be used alone or in combination in the methods of theinvention.

By “antibodies that specifically bind” is intended that the antibodieswill not substantially cross react with another polypeptide. By “notsubstantially cross react” is intended that the antibody or fragment hasa binding affinity for a non-homologous protein which is less than 10%,more preferably less than 5%, and even more preferably less than 1%, ofthe binding affinity for EBI3, p35, or IL35.

The anti-IL35 antibodies disclosed herein and for use in the methods ofthe present invention can be produced using any antibody productionmethod known to those of skill in the art. Thus, polyclonal sera may beprepared by conventional methods. In general, a solution containing theIL35, EBI3 or p35 antigen is first used to immunize a suitable animal,preferably a mouse, rat, rabbit, or goat. Rabbits or goats are preferredfor the preparation of polyclonal sera due to the volume of serumobtainable, and the availability of labeled anti-rabbit and anti-goatantibodies.

Polyclonal sera can be prepared in a transgenic animal, preferably amouse bearing human immunoglobulin loci. In a preferred embodiment, Sf9(Spodoptera frugiperda) cells expressing IL35, EBI3 or p35 are used asthe immunogen. Immunization can also be performed by mixing oremulsifying the antigen-containing solution in saline, preferably in anadjuvant such as Freund's complete adjuvant, and injecting the mixtureor emulsion parenterally (generally subcutaneously or intramuscularly).A dose of 50-200 μg/injection is typically sufficient. Immunization isgenerally boosted 2-6 weeks later with one or more injections of theprotein in saline, preferably using Freund's incomplete adjuvant. Onemay alternatively generate antibodies by in vitro immunization usingmethods known in the art, which for the purposes of this invention isconsidered equivalent to in vivo immunization. Polyclonal antisera areobtained by bleeding the immunized animal into a glass or plasticcontainer, incubating the blood at 25° C. for one hour, followed byincubating at 4° C. for 2-18 hours. The serum is recovered bycentrifugation (e.g., 1,000×g for 10 minutes). About 20-50 ml per bleedmay be obtained from rabbits.

Production of the Sf9 cells is disclosed in U.S. Pat. No. 6,004,552.Briefly, sequences encoding human IL35, EBI3 or p35 are recombined intoa baculovirus using transfer vectors. The plasmids are co-transfectedwith wild-type baculovirus DNA into Sf9 cells. Recombinantbaculovirus-infected Sf9 cells are identified and clonally purified.Recombinant baculovirus-infected Sf9 cells are identified and clonallypurified.

Preferably the antibody is monoclonal in nature. By “monoclonalantibody” is intended an antibody obtained from a population ofsubstantially homogeneous antibodies, that is, the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. The term isnot limited regarding the species or source of the antibody. The termencompasses whole immunoglobulins as well as fragments such as Fab,F(ab′)2, Fv, and others which retain the antigen binding function of theantibody. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site on the target polypeptide. Furthermore,in contrast to conventional (polyclonal) antibody preparations thattypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody is directed against asingle determinant on the antigen. The modifier “monoclonal” indicatesthe character of the antibody as being obtained from a substantiallyhomogeneous population of antibodies, and is not to be construed asrequiring production of the antibody by any particular method. Forexample, the monoclonal antibodies to be used in accordance with thepresent invention may be made by the hybridoma method first described byKohler and Milstein (Nature 256:495-97, 1975), or may be made byrecombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The“monoclonal antibodies” may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal. (Nature 352:624-28, 1991), Marks et al. (J. Mol. Biol. 222:581-97,1991) and U.S. Pat. No. 5,514,548.

By “epitope” is intended the part of an antigenic molecule to which anantibody is produced and to which the antibody will bind. Epitopes cancomprise linear amino acid residues (i.e., residues within the epitopeare arranged sequentially one after another in a linear fashion),nonlinear amino acid residues (referred to herein as “nonlinearepitopes”—these epitopes are not arranged sequentially), or both linearand nonlinear amino acid residues.

As discussed herein, mAbs can be prepared using the method of Kohler andMilstein, or a modification thereof. Typically, a mouse is immunizedwith a solution containing an antigen. Immunization can be performed bymixing or emulsifying the antigen-containing solution in saline,preferably in an adjuvant such as Freund's complete adjuvant, andinjecting the mixture or emulsion parenterally. Any method ofimmunization known in the art may be used to obtain the monoclonalantibodies of the invention. After immunization of the animal, thespleen (and optionally, several large lymph nodes) are removed anddissociated into single cells. The spleen cells may be screened byapplying a cell suspension to a plate or well coated with the antigen ofinterest. The B cells expressing membrane bound immunoglobulin specificfor the antigen bind to the plate and are not rinsed away. Resulting Bcells, or all dissociated spleen cells, are then induced to fuse withmyeloma cells to form hybridomas, and are cultured in a selectivemedium. The resulting cells are plated by serial dilution and areassayed for the production of antibodies that specifically bind theantigen of interest (and that do not bind to unrelated antigens). Theselected mAb-secreting hybridomas are then cultured either in vitro(e.g., in tissue culture bottles or hollow fiber reactors), or in vivo(as ascites in mice).

Where the anti-IL35 antibodies of the invention are to be prepared usingrecombinant DNA methods, the DNA encoding the monoclonal antibodies isreadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Thehybridoma cells described herein serve as a preferred source of suchDNA. Once isolated, the DNA can be placed into expression vectors, whichare then transfected into host cells such as E. coli cells, simian COScells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do nototherwise produce immunoglobulin protein, to obtain the synthesis ofmonoclonal antibodies in the recombinant host cells. Review articles onrecombinant expression in bacteria of DNA encoding an antibody includesSkerra, A. (Curr. Opinion in Immunol. 5:256-62, 1993) and Phickthun, A.(Immunol. Revs. 130:151-88, 1992). Alternatively, antibody can beproduced in a cell line such as a CHO cell line, as disclosed in U.S.Pat. Nos. 5,545,403; 5,545,405 and 5,998,144. Briefly the cell line istransfected with vectors capable of expressing a light chain and a heavychain, respectively. By transfecting the two proteins on separatevectors, chimeric antibodies can be produced. Another advantage is thecorrect glycosylation of the antibody.

Additionally, the term “anti-IL35 antibody” as used herein encompasseschimeric and humanized anti-IL35 antibodies. By “chimeric” antibodies isintended antibodies that are most preferably derived using recombinantdeoxyribonucleic acid techniques and which comprise both human(including immunologically “related” species, e.g., chimpanzee) andnon-human components. Thus, the constant region of the chimeric antibodyis most preferably substantially identical to the constant region of anatural human antibody; the variable region of the chimeric antibody ismost preferably derived from a non-human source and has the desiredantigenic specificity to the IL35 antigen. The non-human source can beany vertebrate source that can be used to generate antibodies to a humanIL35 antigen or material comprising a human IL35 antigen. Such non-humansources include, but are not limited to, rodents (e.g., rabbit, rat,mouse, etc.; see, e.g., U.S. Pat. No. 4,816,567) and non-human primates(e.g., Old World Monkeys, Apes, etc.; see, e.g., U.S. Pat. Nos.5,750,105 and 5,756,096). As used herein, the phrase “immunologicallyactive” when used in reference to chimeric/humanized anti-IL35antibodies means chimeric/humanized antibodies that bind human IL35.

By “humanized” is intended forms of anti-IL35 antibodies that containminimal sequence derived from non-human immunoglobulin sequences. Forthe most part, humanized antibodies are human immunoglobulins (recipientantibody) in which residues from a hypervariable region (also known ascomplementarity determining region or CDR) of the recipient are replacedby residues from a hypervariable region of a non-human species (donorantibody) such as mouse, rat, rabbit, or nonhuman primate having thedesired specificity, affinity, and capacity. The phrase “complementaritydetermining region” refers to amino acid sequences which together definethe binding affinity and specificity of the natural Fv region of anative immunoglobulin binding site. See, for example, Chothia et al. (J.Mol. Biol. 196:901-17, 1987) and Kabat et al. (U.S. Dept. of Health andHuman Services, NIH Publication No. 91-3242, 1991). The phrase “constantregion” refers to the portion of the antibody molecule that conferseffector functions.

Humanization can be essentially performed following the methodsdescribed by Jones et al. (Nature 321:522-25, 1986), Riechmann et al.(Nature 332:323-27, 1988) and Verhoeyen et al. (Science 239:1534-36,1988), by substituting rodent or mutant rodent CDRs or CDR sequences forthe corresponding sequences of a human antibody. See also U.S. Pat. Nos.5,225,539; 5,585,089; 5,693,761; 5,693,762; and 5,859,205. In someinstances, residues within the framework regions of one or more variableregions of the human immunoglobulin are replaced by correspondingnon-human residues (see, for example, U.S. Pat. Nos. 5,585,089;5,693,761; 5,693,762; and 6,180,370). Furthermore, humanized antibodiesmay comprise residues that are not found in the recipient antibody or inthe donor antibody. These modifications are made to further refineantibody performance (e.g., to obtain desired affinity). In general, thehumanized antibody will comprise substantially all of at least one, andtypically two, variable domains, in which all or substantially all ofthe hypervariable regions correspond to those of a non-humanimmunoglobulin and all or substantially all of the framework regions arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin.Accordingly, such “humanized” antibodies may include antibodies whereinsubstantially less than an intact human variable domain has beensubstituted by the corresponding sequence from a non-human species.

Also encompassed by the term “anti-IL35 antibodies” are xenogeneic ormodified anti-IL35 antibodies produced in a non-human mammalian host,more particularly a transgenic mouse, characterized by inactivatedendogenous immunoglobulin loci. In such transgenic animals, competentendogenous genes for the expression of light and heavy subunits of hostimmunoglobulins are rendered non-functional and substituted with theanalogous human immunoglobulin loci. These transgenic animals producehuman antibodies in the substantial absence of light or heavy hostimmunoglobulin subunits. See, for example, U.S. Pat. Nos. 5,877,397 and5,939,598. Preferably, fully human antibodies to IL35 can be obtained byimmunizing transgenic mice. One such mouse is disclosed in U.S. Pat.Nos. 6,075,181; 6,091,001; and 6,114,598.

Fragments of the anti-IL35 antibodies are suitable for use in themethods of the invention so long as they retain the desired affinity ofthe full-length antibody. Thus, a fragment of an anti-IL35 antibody willretain the ability to bind to IL35, EBI3 or p35. Such fragments arecharacterized by properties similar to the corresponding full-lengthanti-IL35 antibody; that is, the fragments will specifically bind IL35,EBI3 or p35. Such fragments are referred to herein as “antigen-binding”fragments.

Suitable antigen-binding fragments of an antibody comprise a portion ofa full-length antibody, generally the antigen-binding or variable regionthereof. Examples of antibody fragments include, but are not limited to,Fab, F(ab′)₂, and Fv fragments and single-chain antibody molecules. By“Fab” is intended a monovalent antigen-binding fragment of animmunoglobulin that is composed of the light chain and part of the heavychain. By F(ab′)₂ is intended a bivalent antigen-binding fragment of animmunoglobulin that contains both light chains and part of both heavychains. By “single-chain Fv” or “sFv” antibody fragments is intendedfragments comprising the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. See, forexample, U.S. Pat. Nos. 4,946,778; 5,260,203; 5,455,030; and 5,856,456.Generally, the Fv polypeptide further comprises a polypeptide linkerbetween the V_(H) and V_(L) domains that enables the sFv to form thedesired structure for antigen binding. For a review of sFv seePluckthun, A. (1994) in The Pharmacology of Monoclonal Antibodies, Vol.113, ed. Rosenburg and Moore (Springer-Verlag, New York), pp. 269-315.

Antibodies or antibody fragments can be isolated from antibody phagelibraries generated using the techniques described in, for example,McCafferty et al. (Nature 348:552-54, 1990) and U.S. Pat. No. 5,514,548.Clackson et al. (Nature 352:624-28, 1991) and Marks et al. (J. Mol.Biol. 222:581-97, 1991) describe the isolation of murine and humanantibodies, respectively, using phage libraries. Subsequent publicationsdescribe the production of high affinity (nM range) human antibodies bychain shuffling (Marks et al., Bio/Technology 10:779-83, 1992), as wellas combinatorial infection and in vivo recombination as a strategy forconstructing very large phage libraries (Waterhouse et al., Nucleic.Acids Res. 21:2265-66, 1993). Thus, these techniques are viablealternatives to traditional monoclonal antibody hybridoma techniques forisolation of monoclonal antibodies.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., J. Biochem.Biophys. Methods 24:107-17, 1992 and Brennan et al., Science 229:81-3,1985). However, these fragments can now be produced directly byrecombinant host cells. For example, the antibody fragments can beisolated from the antibody phage libraries discussed above.Alternatively, Fab fragments can be directly recovered from E. coli andchemically coupled to form F(ab′)₂ fragments (Carter et al.,Bio/Technology 10:163-67, 1992). According to another approach, F(ab′)₂fragments can be isolated directly from recombinant host cell culture.Other techniques for the production of antibody fragments will beapparent to the skilled practitioner.

A representative assay to detect anti-IL35 antibodies specific to theIL35, EBI3 or p35-antigenic epitopes identified herein is a “competitivebinding assay.” Competitive binding assays are serological assays inwhich unknowns are detected and quantitated by their ability to inhibitthe binding of a labeled known ligand to its specific antibody.Antibodies employed in such immunoassays may be labeled or unlabeled.Unlabeled antibodies may be employed in agglutination; labeledantibodies may be employed in a wide variety of assays, employing a widevariety of labels. Detection of the formation of an antibody-antigencomplex between an anti-IL35 antibody and an epitope of interest can befacilitated by attaching a detectable substance to the antibody.Suitable detection means include the use of labels such asradionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,chromogens, enzyme substrates or co-factors, enzyme inhibitors,prosthetic group complexes, free radicals, particles, dyes, and thelike. Such labeled reagents may be used in a variety of well-knownassays, such as radioimmunoassays, enzyme immunoassays, e.g., ELISA,fluorescent immunoassays, and the like. See, for example, U.S. Pat. Nos.3,766,162; 3,791,932; 3,817,837; and 4,233,402.

Small Molecule Screening

The likelihood of an assay identifying an agent that acts as an IL35small molecule inhibitor is increased when the number and types of testagents used in the screening system is increased. Recently, attentionhas focused on the use of combinatorial chemical libraries to assist inthe generation of new small molecule inhibitor leads. A combinatorialchemical library is a collection of diverse chemical compounds generatedby either chemical synthesis or biological synthesis by combining anumber of chemical “building blocks.” For example, a linearcombinatorial chemical library such as a polypeptide library is formedby combining a set of chemical building blocks (amino acids) in everypossible way for a given compound length (i.e., the number of aminoacids in a polypeptide compound). Millions of chemical compounds can besynthesized through such combinatorial mixing of chemical buildingblocks (see, e.g., Gallop et al., 37:1233-50, 1994).

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, for example, peptide libraries (see, e.g., U.S. Pat.No. 5,010,175). Peptide synthesis is by no means the only approachenvisioned and intended for use with the present invention. Otherchemistries for generating chemical diversity libraries can also beused. Such chemistries include: peptoids (see, e.g., WO 91/19735),encoded peptides (see, e.g., WO 93/20242), random bio-oligomers (see,e.g., WO 92/00091), benzodiazepines (see, e.g., U.S. Pat. No.5,288,514), diversomers such as hydantoins, benzodiazepines anddipeptides (see, e.g., Hobbs et al., Proc. Nat. Acad. Sci. USA90:6909-13, 1993), vinylogous polypeptides (see, e.g., Hagihara et al.,J. Amer. Chem. Soc. 114:6568-70, 1992), nonpeptidal peptidomimetics witha β-D-Glucose scaffolding (see, e.g., Hirschmann et al., J. Amer. Chem.Soc. 114:9217-18, 1992), analogous organic syntheses of small compoundlibraries (see, e.g., Chen et al., J. Amer. Chem. Soc. 116:2661-62,1994), oligocarbamates (see, e.g., Cho et al., Science 261:1303-05,1993), and peptidyl phosphonates (see, e.g., Campbell et al., J. Org.Chem. 59:658-60, 1994). In addition, a number of combinatorial librariesare commercially available, as is well known to one of skill in the art.

High throughput techniques are used when screening any of the variouslibraries described herein. As is well known to one of skill in the art,a number of high throughput screening systems are commercially available(e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries, Mentor,Ohio; Beckman Instruments, Inc., Fullerton, Calif.; and PrecisionSystems, Inc., Natick, Mass.). These systems typically automate entireprocedures including all sample and reagent pipetting, liquiddispensing, timed incubations, and final readings of the microplate indetector(s) appropriate for the assay. These configurable systemsprovide high throughput and rapid start up as well as a high degree offlexibility and customization.

Methods of Therapy Using the Compositions of the Invention As disclosedherein, methods of the invention are directed to the use of specificbinding agents for inhibiting T_(R) cell function. Thus, thecompositions are useful for inhibiting T cell function in a subject.EBI3 and p35 pair to form a novel cytokine, IL35, with immunosuppressiveactivity. Interleukin 35 is secreted by CD4⁺Foxp3⁺ T_(R) cells, and maybe secreted by other cells (such as subpopulations of CD8⁺ T cells, γδ Tcells and NK T cells that have regulatory function). It exhibitsimmunoregulatory activity and is required for maximal T_(R) cellfunction. The ability to specifically inhibit IL35 can be used to reduceor block regulatory T cell function. Inhibition may be by antibodies,modified proteins or small molecules that specifically block binding toits receptor or disrupt IL35 chain pairing. As IL35 shares homology insome regions with IL12 and IL27, the inhibitory molecules of theinvention (i.e., IL35-specific antagonists or IL35-specific bindingagents) are designed to recognize and interact with IL35 or its subunitsbut not IL12 or IL27.

The compositions find use in boosting the efficacy of vaccines. Since,T_(R) cells are involved in the induction of tumor antigen tolerance(Mapara and Sykes, J. Clin. Oncology 22:1136-51, 2004), the compositionsare useful for increasing the efficacy of anti-cancer vaccines. ReducingT_(R) cell function can also be beneficial for vaccines that are poorlyimmunogenic; therefore, the compositions can be used with any vaccineincluding vaccines for diphtheria, tetanus, pertussis, polio, measles,mumps, rubella, hepatitis B, Haemophilus influenzae type b, varicella,meningitis, human immunodeficiency virus, tuberculosis, Epstein Barrvirus, malaria, hepatitis E, dengue, rotavirus, herpes, humanpapillomavirus, and cancers

In one embodiment, inhibition of a T_(R) cell function in a subjectincludes administering to the subject a therapeutically effective amountof an IL35-specific binding agent. Administration can begin wheneverinhibition of a T_(R) cell function in a subject is desired, for exampleto prevent or overcome induction of tumor antigen tolerance by T_(R)cells in a subject.

As used herein, “a therapeutically effective amount” of an IL35-specificbinding agent is an amount which, when administered to a subject, issufficient to achieve a desired effect, such as inhibiting a T_(R) cellfunction, in a subject being treated with that composition. For example,this can be the amount of an IL35-specific binding agent useful inpreventing or overcoming induction of tumor antigen tolerance by T_(R)cells in a subject, or the amount required to enhance the efficacy of avaccine (e.g., a cancer vaccine) in a subject. Ideally, atherapeutically effective amount of an IL35-specific binding agent is anamount sufficient to prevent or overcome induction of tumor antigentolerance by T_(R) cells in a subject, or the amount required to enhancethe efficacy of a vaccine (e.g., a cancer vaccine) in a subject, withoutcausing a substantial cytotoxic effect in the subject. The effectiveamount of an IL35-specific binding agent useful for preventing orovercoming induction of tumor antigen tolerance by T_(R) cells in asubject and/or enhancing the efficacy of a vaccine (e.g., a cancervaccine) will depend on the subject being treated, the severity of theaffliction, and the manner of administration of the IL35-specificbinding agent.

In some embodiments a “therapeutically effective amount” or “effectiveamount” (for non-topical administration, such as oral administration, orintravenous or intraperitoneal injection) of a pharmaceuticalcomposition containing an IL35-specific binding agent is from about 0.1to about 200 mg/kg body weight in single or divided doses; for examplefrom about 1 to about 100 mg/kg, from about 2 to about 50 mg/kg, fromabout 3 to about 25 mg/kg, or from about 5 to about 10 mg/kg. Acceptabledosages of the IL35-specific binding agent are, for example, dosagesthat achieve a target tissue concentration similar to that whichproduces the desired effect in vitro. Alternatively, therapeuticallyeffective amounts of an IL35-specific binding agent can be determined byanimal studies. When animal assays are used, a dosage is administered toprovide a target tissue concentration similar to that which has beenshown to be effective in the animal assays. It is recognized that themethod of treatment may comprise a single administration of atherapeutically effective amount or multiple administrations of atherapeutically effective amount of the IL35-specific binding agents ofthe invention.

Any delivery system or treatment regimen that effectively achieves thedesired effect of inhibiting a T_(R) cell function can be used.Accordingly, pharmaceutical compositions including an IL35-specificbinding agent (such as an antibody and/or a small molecule inhibitor)are also described herein. The IL35-specific binding agent is present inthe composition in a therapeutically effective amount.

Formulations for pharmaceutical compositions are well known in the art.For example, Remington's Pharmaceutical Sciences (18^(th) ed.; MackPublishing Company, Eaton, Pa., 1990), describes compositions andformulations suitable for pharmaceutical delivery of one or moreIL35-specific binding agents, such as one or more anti-IL35 antibodiesand/or small molecule inhibitors combined with various pharmaceuticallyacceptable additives, as well as a dispersion base or vehicle. Desiredadditives include, but are not limited to, pH control agents, such asarginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, andthe like. In addition, local anesthetics (e.g., benzyl alcohol),isotonizing agents (e.g., sodium chloride, mannitol, sorbitol),adsorption inhibitors (e.g., Tween 80), solubility enhancing agents(e.g., cyclodextrins and derivatives thereof), stabilizers (e.g., serumalbumin), reducing agents (e.g., glutathione), and preservatives (e.g.,antimicrobials, and antioxidants) can be included.

Therapeutically effective amounts of an IL35-specific binding agent,such as an antibody and/or a small molecule inhibitor, for use in thepresent invention can be administered by any route, including parenteraladministration, for example, intravenous, intraperitoneal,intramuscular, intraperitoneal, intrasternal, or intraarticularinjection, or infusion, or by sublingual, oral, topical, intranasal, ortransmucosal administration, or by pulmonary inhalation. Thepharmaceutical compositions of the present invention can be administeredat about the same dose throughout a treatment period, in an escalatingdose regimen, or in a loading-dose regime (for example, in which theloading dose is about two to five times the maintenance dose). In someembodiments, the dose is varied during the course of a treatment basedon the condition of the subject being treated, the apparent response tothe therapy, and/or other factors as judged by one of ordinary skill inthe art. In some embodiments long-term treatment with a disclosedpharmaceutical composition is contemplated.

In a specific embodiment, it may be desirable to administer atherapeutically effective amount of an IL35-specific binding agent, suchas an antibody and/or a small molecule inhibitor, locally to an area inneed of treatment (e.g., to an area of the body where inhibiting a T_(R)cell function is desired). This can be achieved by, for example, localor regional infusion or perfusion during surgery, topical application,injection, catheter, suppository, or implant (for example, implantsformed from porous, non-porous, or gelatinous materials, includingmembranes, such as sialastic membranes or fibers), and the like. In oneembodiment, administration can be by direct injection at the site (orformer site) of a cancer that is to be treated. In another embodiment,the therapeutically effective amount of an IL35-specific binding agentis delivered in a vesicle, such as liposomes (see, e.g., Langer, Science249:1527-33, 1990 and Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez Berestein and Fidler (eds.), Liss,N.Y., pp. 353-65, 1989).

In yet another embodiment, the therapeutically effective amount of anIL35-specific binding agent, such as an antibody and/or a small moleculeinhibitor, can be delivered in a controlled release system. In oneexample, a pump can be used (see, e.g., Langer, Science 249:1527-33,1990; Sefton, Crit. Rev. Biomed. Eng. 14:201-40, 1987; Buchwald et al.,Surgery 88:507-16, 1980; Saudek et al., N. Engl. J. Med. 321:574-79,1989). In another example, polymeric materials can be used (see, e.g.,Levy et al., Science 228:190-92, 1985; During et al., Ann. Neurol.25:351-56, 1989; Howard et al., J. Neurosurg. 71:105-12, 1989). Othercontrolled release systems, such as those discussed by Langer (Science249:1527-33, 1990), can also be used.

Also provided by the present invention are methods for enhancing theefficacy or immunogenicity of a vaccine in a subject, or overcoming asuppressed immune response to a vaccine in a subject, including (i)administering to the subject a therapeutically effective amount of anIL35-specific binding agent and (ii) administering to the subject avaccine. In one embodiment, the vaccine is a cancer vaccine. In aspecific example, the method further includes administering to thesubject at least one additional therapeutic agent, such as a cytokine, aglucocorticoid, an anthracycline (e.g., doxorubicin or epirubicin), afluoroquinolone (e.g., ciprofloxacin), an antifolate (e.g.,methotrexate), an antimetabolite (e.g., fluorouracil), a topoisomeraseinhibitor (e.g., camptothecin, irinotecan or etoposide), an alkylatingagent (e.g., cyclophosphamide, ifosfamide, mitolactol, or melphalan), anantiandrogen (e.g., flutamide), an antiestrogen (e.g., tamoxifen), aplatinum compound (e.g., cisplatin), a vinca alkaloid (e.g.,vinorelbine, vinblastine or vindesine), or mitotic inhibitor (e.g.,paclitaxel or docetaxel). In some embodiments of the present invention,the amount of the vaccine (and/or the additional therapeutic agent)administered to the subject in the presence of the IL35-specific bindingagent is lower than when the vaccine (and/or the additional therapeuticagent) is administered alone.

By “vaccine” is intended a composition useful for stimulating a specificimmune response (or immunogenic response) in a subject. In someembodiments, the immunogenic response is protective or providesprotective immunity. For example, in the case of a disease-causingorganism the vaccine enables the subject to better resist infection withor disease progression from the organism against which the vaccine isdirected. Alternatively, in the case of a cancer, the vaccinestrengthens the subject's natural defenses against cancers that havealready developed. These types of vaccines may also prevent the furthergrowth of existing cancers, prevent the recurrence of treated cancers,and/or eliminate cancer cells not killed by prior treatments. Withoutbeing bound by theory, it is believed that an immunogenic responsearises from the generation of neutralizing antibodies, T helper cells,or cytotoxic cells of the immune system, or all of the above.

By “enhancing the efficacy” or “enhancing the immunogenicity” withregard to a vaccine is intended improving an outcome, for example, asmeasured by a change in a specific value, such as an increase or adecrease in a particular parameter of an activity of a vaccineassociated with protective immunity. In one embodiment, enhancementrefers to at least a 25%, 50%, 100% or greater than 100% increase in aparticular parameter. In another embodiment, enhancement refers to atleast a 25%, 50%, 100% or greater than 100% decrease in a particularparameter. In one example, enhancement of the efficacy/immunogenicity ofa vaccine refers to an increase in the ability of the vaccine to inhibitor treat disease progression, such as at least a 25%, 50%, 100%, orgreater than 100% increase in the effectiveness of the vaccine for thatpurpose. In a further example, enhancement of theefficacy/immunogenicity of a vaccine refers to an increase in theability of the vaccine to recruit the subject's natural defenses againstcancers that have already developed, such as at least a 25%, 50%, 100%,or greater than 100% increase in the effectiveness of the vaccine forthat purpose.

Similarly, by “overcoming a suppressed immune response” with regard to avaccine is intended improving an outcome, for example, as measured by achange in a specific value, such as a return to a formerly positivevalue in a particular parameter of an activity of a vaccine associatedwith protective immunity. In one embodiment, overcoming refers to atleast a 25%, 50%, 100% or greater than 100% increase in a particularparameter. In one example, overcoming a suppressed immune response to avaccine refers to a renewed ability of the vaccine to inhibit or treatdisease progression, such as at least a 25%, 50%, 100%, or greater than100% renewal in the effectiveness of the vaccine for that purpose. In afurther example, overcoming a suppressed immune response to a vaccinerefers to a renewed ability of the vaccine to recruit the subject'snatural defenses against cancers that have already developed, such as atleast a 25%, 50%, 100%, or greater than 100% renewal in theeffectiveness of the vaccine for that purpose.

As disclosed herein, the present invention provides methods forenhancing the efficacy or immunogenicity of a vaccine in a subject, orovercoming a suppressed immune response to a vaccine in a subject.Representative vaccines include, but are not limited to, vaccinesagainst diphtheria, tetanus, pertussis, polio, measles, mumps, rubella,hepatitis B, Haemophilus influenzae type b, varicella, meningitis, humanimmunodeficiency virus, tuberculosis, Epstein Barr virus, malaria,hepatitis E, dengue, rotavirus, herpes, human papillomavirus, andcancers.

Vaccines of interest include the two vaccines that have been licensed bythe U.S. Food and Drug Administration to prevent virus infections thatcan lead to cancer: the hepatitis B vaccine, which prevents infectionwith the hepatitis B virus, an infectious agent associated with livercancer (MMWR Morb. Mortal. Wkly. Rep. 46:107-09, 1997); and Gardasil™,which prevents infection with the two types of human papillomavirus thattogether cause 70 percent of cervical cancer cases worldwide (Speck andTyring, Skin Therapy Lett. 11:1-3, 2006). Other treatment vaccines ofinterest include therapeutic vaccines for the treatment of cervicalcancer, follicular B cell non-Hodgkin's lymphoma, kidney cancer,cutaneous melanoma, ocular melanoma, prostate cancer, and multiplemyeloma.

The compositions of the invention can be coordinated with treatment withother cancer therapies besides vaccines including chemotherapy,anti-cancer antibody therapy, small molecule-based cancer therapy, andvaccine/immunotherapy-based cancer therapy, and combinations thereof.The compositions of the invention are generally used prior to treatmentwith a vaccine; however, they can be used either prior to, during, orafter treatment of the subject with the other cancer therapy or, in thecase of multiple combination therapies, either prior to, during, orafter treatment of the subject with the other cancer therapies.

As will be understood by one of skill in the art, the methods disclosedherein for enhancing the efficacy or immunogenicity of a cancer vaccinein a subject will be relevant for various types of cancer vaccines,including, but not limited to, antigen/adjuvant vaccines (i.e., one ormore cancer cell antigens combined with an adjuvant), whole cell tumorvaccines (either autologous or allogenic), dendritic cell vaccines(i.e., isolated dendritic cells that are stimulated with the subject'sown cancer antigens and re-injected into the subject), and viral vectorsand DNA vaccines (which use the nucleic acid sequence of a tumor antigento produce a cancer antigen protein).

The immunosuppressive effects of T_(R) cells (as well as the inhibitionthose effects) can be evaluated using many methods well known in theart. In one embodiment, a white blood cell count (WBC) is used todetermine the responsiveness of a subject's immune system. A WBCmeasures the number of white blood cells in a subject. Using methodswell known in the art, the white blood cells in a subject's blood sampleare separated from other blood cells and counted. Normal values of whiteblood cells are about 4,500 to about 10,000 white blood cells/μl. Lowernumbers of white blood cells can be indicative of a state ofimmunosuppression in the subject. In another embodiment,immunosuppression in a subject can be determined by way of a Tlymphocyte count. T lymphocytes are differentiated from other whiteblood cells using standard methods in the art, such as, for example,immunofluorescence or fluorescence activated cell sorting (FACS).Reduced numbers of T cells, or a specific population of T cells (forexample, CD8⁺ T cells) can be used as a measurement ofimmunosuppression. A reduction in the number of T cells, or in aspecific population of T cells, compared to the number of T cells (orthe number of cells in the specific population) prior to a specificevent can be used to indicate that immunosuppression has been induced.

Methods for the isolation and quantitation of T_(R) cells, such asCD4⁺Foxp3⁺ T_(R) cells, and other populations of T cells (e.g., CD8⁺cells), are well known in the art. Typically, labeled antibodiesspecifically directed to one or more cell surface markers are used toidentify and quantify the T-cell population. The antibodies can beconjugated to other compounds including, but not limited to, enzymes,magnetic beads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs. The enzymes that can beconjugated to the antibodies include, but are not limited to, alkalinephosphatase, peroxidase, urease, and β-galactosidase. The fluorochromesthat can be conjugated to the antibodies include, but are not limitedto, fluorescein isothiocyanate (FITC), tetramethylrhodamineisothiocyanate, phycoerythrin (PE), allophycocyanins, and Texas Red. Foradditional fluorochromes that can be conjugated to antibodies seeHaugland, R. P., Handbook of Fluorescent Probes and Research Products,published by Molecular Probes, 9^(th) Edition (2002). The metalcompounds that can be conjugated to the antibodies include, but are notlimited to, ferritin, colloidal gold, and particularly, colloidalsuperparamagnetic beads. The haptens that can be conjugated to theantibodies include, but are not limited to, biotin, digoxigenin,oxazalone, and nitrophenol. The radioactive compounds that can beconjugated or incorporated into the antibodies are known to the art, andinclude, but are not limited to, technetium 99 (⁹⁹Tc), ¹²⁵I, and aminoacids comprising any radionuclides, including, but not limited to, ¹⁴C,³H and ³⁵S.

Fluorescence activated cell sorting can be used to sort cells that areCD4⁺, CD25⁺, both CD4⁺and CD25⁺, or CD8⁺ by contacting the cells with anappropriately labeled antibody. However, other techniques of differingefficacy may be employed to purify and isolate desired populations ofcells. The separation techniques employed should maximize the retentionof viability of the fraction of the cells to be collected. Theparticular technique employed will, of course, depend upon theefficiency of separation, cytotoxicity of the method, the ease and speedof separation, and what equipment and/or technical skill is required.

Additional separation procedures may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents, either joined to a monoclonal antibody or used in conjunctionwith complement, and “panning,” which utilizes a monoclonal antibodyattached to a solid matrix, or another convenient technique. Antibodiesattached to magnetic beads and other solid matrices, such as agarosebeads, polystyrene beads, hollow fiber membranes and plastic Petridishes, allow for direct separation. Cells that are bound by theantibody can be removed from the cell suspension by simply physicallyseparating the solid support from the cell suspension. The exactconditions and duration of incubation of the cells with the solidphase-linked antibodies will depend upon several factors specific to thesystem employed. The selection of appropriate conditions, however, iswell known in the art.

Unbound cells then can be eluted or washed away with physiologic bufferafter sufficient time has been allowed for the cells expressing a markerof interest (e.g., CD4 and/or CD25) to bind to the solid-phase linkedantibodies. The bound cells are then separated from the solid phase byany appropriate method, depending mainly upon the nature of the solidphase and the antibody employed, and quantified using methods well knownin the art. In one example, bound cells separated from the solid phaseare quantified by FACS. Antibodies may be conjugated to biotin, whichthen can be removed with avidin or streptavidin bound to a support, orfluorochromes, which can be used with FACS to enable cell separation andquantitation, as known in the art.

As used herein, the singular terms “a,” “an,” and “the” include pluralreferents unless context clearly indicates otherwise. Similarly, theword “or” is intended to include “and” unless the context clearlyindicates otherwise. It is further to be understood that all base sizesor amino acid sizes, and all molecular weight or molecular mass values,given for nucleic acids or polypeptides are approximate, and areprovided for description. The subject matter of the present disclosureis further illustrated by the following non-limiting examples.

EXPERIMENTAL Example 1 Isolation of Interleukin 35 a Regulatory TCell-Specific Cytokine

An Affymetrix gene analysis was performed for the purpose of identifyinggenes that are preferentially upregulated in or on T_(R) cells. Thisanalysis identified EBI3 as one of those genes. To verify the geneanalysis data, quantitative real-time PCR (qPCR) was used to measureEBI3 mRNA expression. PCR results confirmed the upregulation of EBI3expression in T_(R) cells versus T_(E) cells (FIG. 1). Confirmation ofT_(R)-restricted expression of EBI3 was obtained by additional qPCRanalysis of peripheral CD4⁺CD45RB^(lo)CD25⁺T_(R) cells versus naïveCD4⁺CD45RB^(hi)CD25⁻ T_(E) cells (the standard phenotypic definition forT_(R) and T_(E) cells) purified from C57BL/6 mice, and Foxp3⁺ T_(R)cells versus Foxp3⁻ T_(E) cells sorted from Foxp3^(gfp) knockin mice(Fontenot et al., Immunity 22:329-41, 2005), which express a GFP-Foxp3chimeric protein (FIG. 2A). Literature suggests that neither α or βchains will be secreted alone, but rather, need to pair within the cellto be secreted. Interleukin 27 is a heterodimer of EBI3 and p28, whereasp40 can pair with p19 to yield IL23, or with p35 to yield IL12.Therefore, the expression of p40, EBI3, p35 (IL12a), p28, and p19 inT_(E) cells and T_(R) cells was measured via qPCR to determine putativebinding partners for EBI3 in T_(R) cells. PCR results demonstrated thatp35 was the only IL12 family α chain expressed in T_(R) cells (FIG. 2B).See also, Devergne et al., Proc. Natl. Acad. Sci. USA 94:12041-46, 1997.

The expression of intracellular EBI3 was assessed by flow cytometry inresting T_(R) cells. Using three different EBI3-specific mAbs, restingwild-type T_(R) cells, but not wild-type T_(E) or EBI3^(−/−) T_(R)cells, were shown to express intracellular EBI3. Finally, immunoblotanalysis clearly revealed the coimmunoprecipitation of EBI3 with IL12ain supernatants from resting T_(R), but not T_(E) cells or EBI3^(−/−)T_(R) cells (FIG. 2C). Taken together, these data demonstrate thepreferential secretion of a novel EBI3/IL12a heterodimeric cytokine byT_(R) cells amongst CD4⁺ T cell populations.

Given that T_(R) cells require activation through their TCR in order toexert their suppressive activity (Thornton et al., J. Exp. Med.188:287-96, 1998; Thornton et al., J. Immunol. 164:183-90, 2000;Takahashi et al., Int. Immunol. 10:1969-80, 1998), an assessment of howEBI3 and IL12a mRNA levels were altered following T_(R) cell activationin the absence or presence of T_(E) cells was made. Both EBI3 and IL12amRNA were significantly reduced following anti-CD3 stimulation, butdramatically upregulated (234- and 740-fold, respectively) in T_(R)cells recovered from an in vitro T_(R) assay, and thus in the process ofactive suppression (FIG. 2D). Indeed, the increase in IL12a mRNA farexceeded that observed in activated macrophages. These data demonstratethat a novel EBI3/IL12a heterodimeric cytokine is produced by T_(R)cells, which is potentiated during active suppression of T_(E) cells.

The discrete, differential expression of EBI3 in T_(R) versus T_(E)cells suggests that its expression may be controlled by transcriptionalprocesses that regulate T_(R) development and function. Indeed, EBI3expression was concordant with Foxp3, which is required for T_(R)development (Zheng et al., Nat. Immunol. 8:457-62, 2007). EBI3 mRNA waspresent in CD4⁺Foxp3⁺ thymocytes but essentially absent in CD4⁺CD8⁺ andCD4⁺Foxp3⁻ thymocytes. To determine if EBI3 is a downstream target ofFoxp3, purified T_(E) cells were transduced with retroviral vectorsencoding Foxp3 plus GFP or GFP alone. Foxp3-transduced T_(E) cellsexhibited considerably elevated EBI3 transcript levels compared with theGFP alone controls, while Foxp3 induced limited expression of IL12a mRNA(FIG. 2E). These data provide a mechanistic basis for the restrictedsecretion of the EBI3/IL12a heterodimer by T_(R) cells, with EBI3 beinga downstream target of Foxp3.

Example 2 Interleukin 35 is Required for Optimal T_(R) Cell Function

Neither EBI3^(−/−) nor IL12a^(−/−) mice have any overt autoimmunity orinflammatory disease (Boirivant et al., J. Exp. Med. 188:1929-39, 1998;Mattner et al., Eur. J. Immunol. 26:1553-59, 1996). Indeed, thepercentage of T_(R) cells in these mice and their Foxp3 expression iscomparable to wild-type mice. This raises the possibility that theconsequence of lacking a negative regulatory EBI3/IL12a cytokine may benegated by the lack of the proinflammatory cytokines IL27 and IL12 inthe EBI3^(−/−) and IL12a^(−/−) mice, respectively. Indeed, whenchallenged, EBI3^(−/−) mice are more susceptible to leishmaniasis (Zahnet al., Eur. J. Immunol. 35:1106-12, 2005). Likewise, IL12a^(−/−),distinct from IL12b^(−/−) (p40) mice, are more susceptible toHelicobacter-induced colitis (Kullberg et al., J. Exp. Med. 203:2485-94,2006), Leishmania major infection (Mattner et al., Eur. J. Immunol.26:1553-59, 1996), experimental autoimmune encephalomyelitis (Gran etal., J. Immunol. 169:7104-10, 2002; Becher et al., J. Clin. Invest.110:493-97, 2002), and collagen-induced arthritis (Murphy et al., J.Exp. Med. 198:1951-57, 2003).

To determine whether the loss of EBI3 or IL12a expression would havefunctional implications for T_(R) cells, T_(E) cells and T_(R) cellswere isolated from wild-type, EBI3^(−/−) and IL12a^(−/−) mice (Boirivantet al., J. Exp. Med. 188:1929-39, 1998; Mattner et al., Eur. J. Immunol.26:1553-59, 1996). An in vitro T_(R) cell assay was performed todetermine whether T_(R) cells lacking EBI3 or IL12a could suppress T_(E)cell proliferation. Wild-type T_(R) cells could suppress proliferationof T_(E) cells in a dose dependent manner. In contrast, both EBI3^(−/−)and IL12a^(−/−) T_(R) cells were less capable of suppressing T_(E) cellproliferation, showing that EBI3 and IL12a are required for optimalT_(R) cell function (FIGS. 3A & 3B).

To determine EBI3^(−/−) and IL12a^(−/−) T_(R) cell function in vivo,their ability to control the homeostatic expansion of T_(E) cells wasevaluated. In vivo, T_(R) cells have been shown to control thehomeostatic expansion of T_(E) cells in a lymphopenic, RAG1^(−/−)environment (Annacker et al., Immunol. Rev. 182:5-17, 2001; Annacker etal., J. Immunol. 164:3573-80, 2000; Workman et al., J. Immunol.174:688-95, 2004). Therefore, to determine whether the expression ofEBI3 and IL12a influenced the ability of T_(R) cells to controlhomeostatic expansion, purified wild-type T_(E) cells either alone, orin the presence of wild-type, EBI3^(−/−) or IL12a^(−/−) T_(R) cells,were adoptively transferred into RAG1^(−/−) mice. As RAG1^(−/−) micelack T and B cells, expansion of adoptively transferred T cellsrepresent the only T cell population present in these mice. Splenic Tcell numbers were determined 7-10 days post-transfer. In the presence ofwild-type T_(R) cells, T_(E) cell expansion was significantly reduced,while minimal reduction in wild-type T_(E) cell expansion was observedin the presence of either EBI3^(−/−) or IL12a^(−/−) T_(R) cells (FIG.3C).

T_(R) cells have also been shown to control colitis in mice, resemblingIBD, that is initiated experimentally by transferring naïve T cells intoRAG1^(−/−) recipients (Izcue et al., Immunol. Rev. 212:256-71, 2006). Inthese experiments, severity of disease is monitored clinically, byweight loss, and histologically, utilizing a semi-quantitative gradingscheme to score pathology. Recovery from disease, marked by weight gainand decreased histopathology, is observed only in mice that receivepurified T_(R) cells approximately four weeks after the initial T_(E)cell transfer (Mottet et al., J. Immunol. 170:3939-43, 2003).

This recovery model of IBD was chosen to test the functionality ofEBI3^(−/−) and IL12a^(−/−) T_(R) cells in vivo. After wild-typeT_(E)-recipient RAG1^(−/−) mice developed clinical symptoms of IBD(approximately 4 weeks), they received wild-type, EBI3^(−/−) orIL12a^(−/−) T_(R) cells and were monitored daily. Wild-typeT_(R)-recipient mice were noticeably healthier within 5-7 days, hadrestored appetite, and resumed weight gain (FIG. 4A). However,EBI3^(−/−) and IL12a^(−/−) T_(R) recipients continued to lose weight,with some mice dying within the first 10 days post-T_(R) cell transfer.After 4 weeks (8 weeks after initial T_(E) cell transfer), histologicalanalysis was performed to assess the extent of recovery. Severe IBDpathology including loss of goblet cells and mucus secretion, mucosalhyperplasia, extensive ulceration, marked transmural lymphohistiocyticinflammation, extensive infiltration of CD3⁺ T cells, and effacement ofthe normal architecture by the inflammatory infiltrate was observed inthe non-T_(R) recipients. In wild-type T_(R) recipients, there wassubstantial reduction of the mean pathology score, significantly reducedinflammation, reduced CD3⁺ T cell infiltration, and regeneration ofgoblet cells, and mucus secretion. In contrast, EBI3^(−/−) andIL12a^(−/−) T_(R) recipients had only an approximately 50% reduction inthe pathology score, as defined by goblet cell destruction, mucosalhyperplasia and cellular infiltration (FIG. 4B). Thus, the slightlyimproved histological score was insufficient to mediate weight gain andrecovery from colitis. Similarly, EBI3^(−/−) and IL12a^(−/−) T_(R) wereunable to reduce colitis and weight loss to the same extent as wild typeT_(R) cells in a traditional co-transfer model of IBD. These resultsdemonstrate that EBI3 and IL12a are required by T_(R) cells for maximalregulatory activity in vitro and in vivo.

Example 3 Both EBI3 and IL12a are Required for the Generation ofInterleukin 35

Several studies have shown that ectopic expression of Foxp3 or theregulatory protein LAG-3 can confer regulatory activity on naïve T_(E)cells (Hori and Sakaguchi, Science 299:1057-61, 2003; Fontenot et al.,Nat. Immunol. 4:330-36, 2003; Huang et al., Immunity 21:503-13, 2004).As the qPCR data indicated that EBI3+IL12a is a functional heterodimerimportant to T_(R) cell function, EBI3+IL12a was ectopically expressedto see if its expression could confer regulatory activity tonon-regulatory T cells. Naïve T_(E) cells from hemagglutinin-specificclone 6.5 TCR transgenic mice were transduced with EBI3, IL12a,EBI3+IL12a, or vector alone to assess the impact of expressing theseproteins on cellular function. With ectopic expression of EBI3+IL12a,but not with either protein alone, transduced T_(E) cells gained T_(R)cell function as measured by their ability to inhibit proliferation ofnaïve T cells (FIG. 5A). Recombinant IL35 derived from 3T3 cells or 293Tcells also inhibited T cell proliferation. The observation thatnon-regulatory T cells gain regulatory activity by the expression ofEBI3+IL12a, but not independently, demonstrates that both EBI3 and IL12aare required for the generation of this regulatory cytokine.

Purified T_(E) cells from the clone 6.5 TCR transgenic mice were alsotransduced with retroviral vectors encoding the expression of GFP alone,or GFP plus either Foxp3, EBI3, IL12a or “native” IL35 (i.e.,EBI3-2A-IL12a—stoichiometric, bicistronic expression of EBI3 and IL12ain a single vector; Szymczak-Workman et al. in Gene Transfer: Deliveryand Expression, Friedmann and Rossi (eds.), Cold Spring HarborLaboratory Press, N.Y., pp. 137-47, 2006; Szymczak and Vignali, Exp.Opin. Biol. Ther. 5:627-38, 2005; Hoist et al., Nature Methods 3:191-97,2006). T cell transductants were sorted for GFP equivalency andco-cultured with naïve, wild-type T_(E) cells in an antigen-drivenproliferation assay to determine if these proteins bestowed regulatorypotential. The results confirmed that T cells expressing IL35, but noteither chain alone, suppressed T_(E) cell proliferation in a titratablefashion, to a level that was approximately two thirds of the regulatoryactivity observed with the Foxp3-transduced T cells (FIG. 5B).

Given that IL35 is secreted by T_(R) cells and forced expression confersregulatory activity on an otherwise non-regulatory T cell, an assessmentwas made as to whether recombinant IL35 could directly inhibit T_(E)cell proliferation. HEK293T cells (human embryonic kidney) weretransfected with plasmids encoding expression of either “native” IL35(EBI3-2A-IL12a) or “single chain” IL35 (i.e., EBI3 and IL12a expressedas a single chain protein; Hisada et al., Cancer Res. 64:1152-56, 2004).Empty vector, EBI3 alone and IL12a alone controls were also generated.Recombinant IL35 was then assessed to determine if it could suppress theproliferation of T_(E) cells stimulated with anti-CD3/CD28-coatedmicrobeads. Media containing either form of recombinant IL35, but notany of the three controls, potently suppressed T_(E) cell proliferationin a titratable fashion (FIG. 5C). Co-culture with irradiated HEK293Tcell transfectants gave identical results. These data demonstrate thatsoluble, recombinant IL35 alone is sufficient to suppress T cellproliferation.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1.-25. (canceled)
 26. A method of culturing a population of T effectorcells comprising: a. isolating a population of T effector cells; b.activating said population of T effector cells; and, c. culturing saidactivated population of T effector cells with ectopically expressedIL35, wherein said ectopically expressed IL35 is present in an amountsufficient to suppress T effector cell proliferation.
 27. The method ofclaim 26, wherein the IL-35 polypeptide comprises an EBI3 subunit and aP35 subunit which are not covalently linked to one another.
 28. A cellcultured by the method of claim
 26. 29. A cell cultured by the method ofclaim 27.